Many couples throughout the world have the unfortunate problem of being unable to produce progeny, or, in other words, are infertile. Interestingly, of those infertile couples, approximately 50% are affected because of a problem with the male. A technique that has been found to be somewhat effective with some such couples is in vitro fertilization (IVF), also referred to as human assisted reproductive technology (ART). Although ART has made great strides during the past decade, it is still a relatively inefficient technology. The mean clinical pregnancy rate (CPR) in the U.S. was 35% according to data for 1998 (SART Report, 2000).
During their transit through the epididymis, spermatozoa undergo biochemical and morphological changes to acquire motility and the ability to fertilize an oocyte in vivo. Much of the biochemical changes that occur during this maturation process involve plasma membrane molecules. These changes are believed to be of critical importance because the sperm surface is involved in intercellular and intracellular aspects of fertilization.
A functional marker of sperm fertilizing capacity and for success of in vitro fertilization is sperm hyaluronidase. Sperm hyaluronidase is now known to be the sperm membrane protein called the Sperm Adhesion Molecule 1 (SPAM 1 or PH-20). The molecule is the most widely conserved mammalian sperm membrane protein and is known to play at least three major roles in fertilization.
Via its neutral insoluble hyaluronidase activity, SPAM1 is primarily responsible for the dispersion of the cumulus matrix which forms the first formidable barrier surrounding the oocyte. The molecule also has a hyaluronic acid (HA) receptor which plays a role in the intracellular signaling that is responsible for the acrosome reaction. This reaction releases from the sperm soluble hyaluronidase with an acidic catalytic activity which plays a role in penetrating the zona pellucida and the dispersion of hyaluronic acid in the perivitelline space, the other barriers surrounding the egg. Finally, the adhesion domain found in the C-terminus of the molecule is responsible for the secondary binding of the sperm to the zona pellucida, after the acrosome reaction has occurred. This secondary binding, along with the acidic hyaluronidase activity, is necessary to assist the sperm in navigating the zona pellucida.
Unlike somatic ubiquitous hyaluronidases that are active only at acidic pH, SPAM 1 has hyaluronidase activity at both neutral and acidic pHs, which arise from two different regions within the hyaluronidase domain. The neutral enzyme activity, which is predominantly in the insoluble membrane-bound SPAM 1, is necessary for sperm to penetrate the (HA)-rich extracellular matrix of the cumulus cells surrounding the oocyte. The acidic enzyme activity is present in soluble SPAM 1 that is generated during the acrosome reaction (AR), after cleavage at its carboxy terminus. In primate sperm where membrane-bound SPAM 1 is a 64-kDa protein, the proteolytically cleaved secretory form that is released after the AR is 53 kDa.
Originally, SPAM 1 was thought to be testis-specific. However, it has now been documented that SPAM 1 is also expressed in all three regions of the epididymis (i.e. caput, corpus and cauda) as well as the uterine epithelium during estrus/menstrual cycle. This indicates that it is involved in both sperm maturation and storage in the male and also the final sperm maturation that occurs in the female (capacitation).
To date, SPAM 1 has been found to be expressed in the epididymis of at least four mammalian species (mice, rats, macaques, and humans). This conservation of the expression of SPAM1 in the epididymis suggests that it plays an important functional role in mammalian sperm maturation, the primary function of the epididymis. Epididymal SPAM1 has been characterized in the mouse where it has been shown to be released in vitro as well as in vivo, demonstrating that it is a secretory protein. Importantly, SPAM1 is secreted in the epididymal luminal fluid in both a soluble and a membrane-bound vesicular form; the latter has been shown to possess its lipid glycosyl phosphatidyl inositol (GPI) anchor in an intact form. An intact GPI anchor on epididymal SPAM1 allows the protein to bind to the sperm plasma membrane during their transit in the epididymis to enhance the level of pre-existing molecules obtained from the testes, and thus increase the fertilizing competence of sperm.
Because the SPAM 1 gene is widely conserved among mammals and because its dual pattern of expression in the male (i.e. in the testis and epididymis) is conserved in at least the four species mentioned above, it is likely that the secretion of epididymal SPAM1 is also conserved. This is further supported by evidence that SPAM1 is conserved among mammals in the female uterine epithelium where it is expressed as a secretory protein as well. Accordingly, human SPAM 1 is expected to be secreted in the epididymis, where it has an important, putative role in sperm maturation.
Further, it is believed that SPAM1 is produced independently in the testis and epididymis thus revealing that testicular SPAM1 and epididymal SPAM1 are different glycoforms and that the latter shares identical glycans with caudal sperm. As such, it seems likely that epididymal SPAM1 is a unique isoform, not a redundant protein, further supporting the position that it plays a specific role in sperm maturation in humans where SPAM1 is the only reproductive hyaluronidase in the cluster on chromosome 7q31.
Recently, a soluble recombinant human SPAM1/PH-20 hyaluronidase (rHPH-20) was expressed and purified with high enzyme activity (approximately 75,000 Units/mg) from a stable transfected CHO cell line developed in a chemically defined, protein-free, tissue culture system (Kennedy C., Kunda A., Bleil J. D., Frost G. I., Bookbinder L. H. Dispersion of the cumulus matrix with a highly purified recombinant human hyaluronidase (RHUPH20), Hyaluronan 2003, Oct. 11-16, Poster # 133, The Cleveland Clinic and Matrix biology Institute). While this product has superior enzymatic activity compared to slaughter-house-derived hyaluronidase preparations, it has not been engineered to assist the sperm protein in performing its receptor activity, necessary for intracellular signaling, or its adhesion activity and the ensuing acidic enzymatic penetration of the zona pellucida. In addition, it is important to also note that the lipid anchor was removed from that preparation of the protein, thus eliminating the ability of the molecule to chemically attach to the sperm membrane and thus function in other important aspects other than its enzymatic hyaluronidase activity.
There is therefore a need for an in vitro process for presenting SPAM1 to sperm such that it binds at its in situ location on sperm thus increasing the sperms' capability of performing the additional steps required for fertilization.